Project Summary: Glaucoma is a major blinding disease, with elevated intraocular pressure (IOP) as a crucial risk factor. Although various medications, stents, lasers, and surgeries are currently used to treat glaucoma patients, some are invasive, others often exhibit limited effectiveness or become so over time and patient compliance remains a major issue. The normal trabecular meshwork (TM) facilitates intraocular pressure (IOP) homeostasis, i.e. responds to pressure disbalances and adjusts the outflow resistance correctively, while the glaucomatous TM cannot. Glaucomatous eyes also have reduced numbers of TM cells. Our previous work demonstrated that TM cellularity reduction in a model system compromised IOP homeostatic response. Our long-term goal is to restore IOP homeostatic capability by transplanting autologous, patient-derived human induced pluripotent stem cells (iPSCs) or human mesenchymal stem cells (hMSCs) to treat glaucomatous cell loss. Previously we transplanted either of these two cell types, differentiated to TM-like cells, into TM in a human cell-depletion model for glaucoma. These differentiated stem cells restored IOP homeostasis by an undetermined mechanism. The goal of this proposal is to establish what that mechanism is and to evaluate conditions that may optimize moving eventually toward clinical trials in a new era of regenerative medicine. Our central working hypothesis is that the transplanted differentiated stem cells integrate into the tissue and are restoring function directly. Alternative possibilities include the stem cells triggering endogenous TM cell division or that a stem cell-produced factor is responsible for the effect. In Specific Aim 1, we will determine the mechanism of stem cell restoration of IOP homeostasis one week after transplantation. We will do this with both TM-like iPSCs and TM-like hMSCs. In Specific Aim 2, we will extend the studies, focusing on the mechanism(s) found to be important in Aim 1, to a longer duration and will compare TM-like iPSCs to TM-like hMSCs for efficacy, efficiency and longevity of IOP homeostatic restoration. In Specific Aim 3, we will determine the relative efficacy of TM-like iPSCs and TM-like hMSCs in restoring IOP homeostatic capability to glaucomatous anterior segments. Completion of these studies is a necessary step to inform any future clinical stem cell therapies for glaucomatous IOP homeostatic restoration.